Download Determination of activity and Michaelis constant of LD in biological

Biochemistry
2nd year
DETERMINATION OF ACTIVITY AND MICHAELIS CONSTANT OF LDH IN BIOLOGICAL
MATERIAL
Attention: For a successful completion of this practical training is necessary to have 1 page of millimeter paper, ruler and
calculator for each group (2 students)! Practical training follows the lecture “Enzymology” and seminar dedicated to the
“Enzymology and enzyme kinetics”. The necessary part for your procedure in this practical training is on pages 3-6.
1. LACTATE DEHYDROGENASE: BIOCHEMISTRY AND CLINICAL MEDICINE
Lactate dehydrogenase (EC 1.1.1.27) is an
enzyme that can be found in almost all tissues
in human body. It catalyses the pyruvate - Llactate change including the oxidation of NADH
to NAD+. This enzyme consists of two types of
polypeptide chains H and M, which form
tetramer with molecular weight 140 kDa. There
are 5 isoenzymes of LDH with the following
composition M4, M3H, M2H2, MH3, and H4. In
aerobic conditions pyruvate is converted to
acetyl-CoA which enters Krebs cycle. In
anaerobic conditions pyruvate is changed to
lactate. The concentration of lactate in cytosol
depends on NADH/NAD+ ratio. During tissue
hypoxia the reoxidation of NADH and FADH2 in
respiratory chain in mitochondria is decreased.
Decreased generation of ATP and energetic
deficit can lead to a decreased function of the
whole cell. The ratio NADH/NAD+ in cytosol is
increased and it is related to the increase of
lactate concentration as in anaerobic
metabolism.
Disorder
of
metabolism,
hypoperfusion of tissues and deficiency of
thiamine can indicate hyperlactemia or lactic
acidosis. The use of different medicaments,
together with combination of pathological
changes of vascular system, liver or kidneys,
leads in elderly people to increased risk of lactic
acidosis with fatal prognosis.
Case report:
A 62 year old woman is admitted to hospital
after she has been suffering from dyspnea and
fuzziness for two hours. She denies chest pain.
However, she feels a slight pressure near heart.
She has been taking medications for diabetes
type II, hypertension and hyperlipoproteinemia
for many years. She takes 3 g of metformin in
combination with 30 mg gliclazide (diabetes
therapy) every day. She takes 20 mg of
atorvastatin every day (hyperlipoproteinemia
therapy), as well as nitroglycerine and
acetylsalicylic acid 100 mg for the treatment of
stable angina pectoris. She is an ex-smoker
and has not smoked for 5 years. She used to
smoke 8 cigarettes per day. She denies alcohol
abuse.
Basic biochemical and acid-base balance
analyses were done. The results indicated the
presence of lactic acidosis (Table 1).
Urine analysis: glucose +, other analytes were
negative. The oxygen mask was immediately
applied and the patient was treated with
bicarbonate and the lost fluid was replenished.
Renal function was satisfactory and the patient
did not undergo hemodialysis. Cardiological
intervention was undertaken by reperfusion.
Further examination confirmed anteroseptal
infarction as a result of damaged left coronary
artery.
Parameter
Value
Reference range
Na+
135 mmol/L
136-146 mmol/L
K+
3.3 mmol/L
3.8-5.0 mmol/L
Glucose
10 mmol/L
3.9-5.6 mmol/L
pH
6.8
7.36-7.44
paCO2
4.32 kPa
4.40-5.73 kPa
p a O2
9.07 kPa
10.4-14.3 kPa
HCO35 mmol/L
22-26 mmol/L
Lactate
18 mmol/L
0.5-2.0 mmol/L
Creatinine
90 µmol/L
44-104 µmol/L
Table 1: Selected results of biochemical examination.
On admission, the patient had bradycardia
(45/min) and deep and fast breathing (30/min).
She was pale and sweating a lot. Her body
temperature was 36.2 °C; blood pressure was
100/60 mmHg. Electrocardiogram showed the
presence of the elevation of ST segment and
the blockage of the left Tawara-branch block.
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Biochemical aspects of cardiac ischemia:
Metformin is a per oral antidiabetic drug and it
belongs to the class of biguanides. It is the
medicament that is prescribed to obese patients
with diabetes type II and with normal renal
function. Metformin decreases both basal and
postprandial concentration of plasma glucose. It
decreases gluconeogenesis and glycogenolysis
in liver and enhances recapturing of glucose at
periphery, as well.
in the decrease of ATP production in
mitochondria, inhibition of Krebs cycle, and
increase of NADH/NAD+ ratio with conversion
to
anaerobic
metabolism.
Increasing
concentration of lactate in cells and decreasing
pH result in the inhibition of glycolytic enzymes.
Production of ATP via anaerobic metabolism is
weakened and the resulting accumulation of
toxic products leads to myocardial necrosis.
Development of massive lactic acidosis in the
acute myocardial infarction and metformin
therapy is observed. Ionic and acid-base
homeostasis is impaired and this affects the
vascular system; therefore, the risk of patient’s
death is increased. Lactate dehydrogenase is
an enzyme used in diagnosis of heart ischemia
(mainly LDH1 and LDH2). LDH1/LDH2 ratio
higher than one suggests increased probability
of myocardial infarction. Other significant
markers of myocardial infarction are, among
others, myoglobin, troponin I or T, and CKMBmass. LDH is also a tumour marker.
Lactic acidosis is a rare but very serious
complication of metformin therapy. Metformin is
contraindicated in patients who suffer from
renal insufficiency, serious infection or a
condition associated with tissue hypoxia.
Patients with cardiac disorder (stabile angina
pectoris) can suffer from insufficient blood
supply in myocardium if they do physical
exercises. Decreased supply of oxygen can
cause ischemia and a subsequent pain. Heart
muscle has a high oxygen demand. Temporary
ischemia and decreased oxygen supply result
2. GENERAL PRINCIPLES OF PROTEIN ISOLATION
For isolation of the required protein, the choice of right material plays a crucial role. The material should
be easily available and should contain the required protein in sufficient quantity. Strategy of isolation
depends on the localisation of the protein in cell. The main aim of the first step is to dissolve the protein
and discard all insoluble parts. For isolation of intracellular proteins it is important to homogenise the
cells and transfer the cell material to appropriate buffer.
2.1 Homogenisation
The type of homogenisation is dependent on the character of used material. For animal tissues it is
possible to use the meat grinder. Homogenisation can be carried out by mixer or by homogeniser, e.g.
Potter and Elvehjem.
2.2 Extraction
Proteins from homogenised animal tissue are then extracted into 2 – 5 times volume of buffer. This
buffer should have sufficient buffer capacity. Extraction should be carried out in the solution with stable
pH. It is recommended to work at lower temperatures, since at these temperatures proteins are more
stable (4 °C – the denaturation process is slow).
2.3 Precipitation
Proteins are then precipitated by the addition of neutral salt to the water solution. Protein molecules
without solvation cover form aggregates when the salt is added. Eventually, these protein aggregates do
precipitate. By applying the correct concentration of the salt in solution we can precipitate particular type
of proteins due to the different water solubility of proteins. Salting effect is higher if pH equals pI (pH =
pI), and the resulting charge of protein is therefore minimal. This means that the solubility decreases at
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Biochemistry
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this pH. The most commonly used salt for this process is ammonium sulphate. It is very important to add
salt to solution gradually by low amounts with continuous mixing.
2.4 Centrifugation
Centrifugation is often faster and more suitable process than filtration. Sometimes it can lead to better
separation of solid and liquid phase even in one step.
3. ISOLATION OF LACTATE DEHYDROGENASE FROM HEART MUSCLE
Enzyme is extracted into phosphate buffer and purified by ammonium sulphate. Catalytic activity of LDH
is proportional to the produced pyruvate per time unit under standard conditions. Generated pyruvate
reacts with 2,4-dinitrophenylhydrazine, forming coloured hydrazone, which can be measured
photometrically.
OH
O
H3C
LDH
H3C
O
O
HO
NAD
+
NADH + H
+
HO
Reaction where the coloured hydrazone is generated:
CH3
H2N
NH
O
O
O
N
+
N
H3C
O
-
O
+
OH
HO
HN
O
N
+
-
O
+
N
-
O
O
+
O
-
N
O
Procedure:
1.
2.
3.
Cut 30 – 40 g of chicken heart into small pieces and homogenise in mixer with 100 mL of 0.2 M
phosphate buffer with 1 mM EDTA. Extract homogenate for 15 min and stir occasionally.
Centrifuge at 4000 RPM at 4 °C for 10 min. Supernatant is called rough extract.
Add solid ammonium sulphate (until 35% saturation is achieved) into the rough extract with
continuous mixing. Incubate suspension for 15 min and then centrifuge according to previously
described conditions. Add ammonium sulphate up to 60% saturation into the supernatant and
then centrifuge again.
Re-suspend sediment in small amount of 0.1 M TRIS buffer (maximal 5 mL).
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Biochemistry
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4. DETERMINATION OF CATALYTIC ACTIVITY AND MICHAELIS CONSTANT OF LDH WITH
LACTATE AS A SUBSTRATE
After the lecturer’s permission, pipette ………. µL of prepared fraction of LDH in tube and add ……….
µL of 0.1 M TRIS buffer (final dilution: …………..). Diluted sample keep on the ice.
4.1 Preparation of sample for activity determination and estimation of Michaelis constant for
LDH with lactate as a substrate
Pipette solutions to 6 tubes as follows:
Tube
1 (Blank)
2
3
4
5
6
0.9 M Lactate [mL]
3 mM NAD+ [mL]
Dist. water [mL]
0.1 M TRIS buffer [mL]
0.10
0.01
0.02
0.04
0.05
0.10
0.15
0.15
0.15
0.15
0.15
0.15
0.09
0.08
0.06
0.05
-
0.1
0.1
0.1
0.1
0.1
0.1
Table 2
1.
2.
3.
4.
Put the tubes with all solutions into the water bath and incubate for 5 min at 37 °C.
Add 0.1 mL of isolated enzyme into tubes 2-6 exactly in 10 s interval and incubate in the water
bath for another 5 min at 37 °C.
After incubation, pipette again exactly in 10 s interval to all tubes 0.25 mL of 2,4dinitrophenylhydrazine. Let them stay for 10 min at laboratory temperature.
Finally, add to all tubes 2.5 mL of NaOH and after 10 min measure absorbance against blank at
505 nm.1
5. EXPERIMENTAL DATA PROCESSING
5.1 Determination of LDH activity
For calculation of LDH activity, use absorbance value measured in tube 6 against blank. Activity will be
expressed as the concentration of formed pyruvate per minute in one milliliter of the sample.
LDH activity

A
 378,6  F
t
2
LDH activity: …………………………………….. µmol.min-1.mL-1
Calculation:
In laboratory practise, reduction of produced pyruvate to lactate with NADH as a cofactor is used. NADH is oxidized to
NAD+ in redox reaction. This oxidation induces change in kinetic mode caused by decrease of absorbance at 340 nm.
2 F is a dilution factor. It determines how many times you diluted the prepared enzyme fraction before the sample was
analysed (see the part 4 of this procedure).
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5.2 Estimation of Michaelis constant for lactate:
Use Lineweaver-Burk plot (double-reciprocal) to determine Michaelis constant for lactate. To axis x plot
1/[lactate] and to axis y plot 1/A (use millimeter paper):
Tube
c(lactate)
1/[lactate]
A
1/A
1 (Blank)
-
-
-
-
2
3
4
5
6
Table 3
Calculate the value of Michaelis constant for lactate from the intersection point of the straight line and
the axis x.
KM (lactate) = ………………
Calculation:
Discussion:
1. Interpret the pathological results from biochemical analysis that was done for this patient.
2. Try to think why lactic acidosis is dangerous of the organism? What are the other causes of
lactic acidosis?
3. Explain the term Michaelis constant and its importance.
4. Try to explain why the increasing order of lactate concentration and the constant NAD +
concentration were used for determination of KM for lactate? What would you do if you were to
determine KM for NAD+?
5. Why is it good to add EDTA into the buffer used for homogenisation?
Conclusion:
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Biochemistry
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APPENDIX 1: DOUBLE-RECIPROCAL PLOT
Enzymes are protein catalysts that accelerate chemical reaction in an organism. Enzymes accelerate
the rate of reaching equilibrium between default substances (substrates) and products only by decrease
of activation energy. Enzymes divide the reaction into several steps. Final composition of balance
mixture is not affected by enzymes. Specific feature of enzymatic reaction is saturation by substrate.
Michaelis and Menten described the simplest mechanism of enzymatic reaction. Enzymatic reaction
occurs by following scheme: enzyme (E) reacts at first with substrate (S) and generates enzymesubstrate complex (ES) which subsequently splits to enzyme and product (P).
k1
E+ S
k -1
ES
k2
E+P
For initial velocity (v) of the reaction, which is in steady state (concentration of ES complex is constant)
and in which the concentration of substrate is much higher than the concentration of enzyme, it applies
that:
𝑣=
𝑉𝑚𝑎𝑥 . 𝑆
𝐾𝑀 + 𝑆
where Vmax is maximal velocity of reaction and KM is the Michaelis constant, which characterises affinity
of enzyme to substrate. Michaelis and Menten equation is hyperbolic function. It suits very well for
experimental finding for many enzymes. Through simple mathematic operations it is possible to linearize
the hyperbola. This adjustment was suggested by Lineweaver and Burk:
1
𝐾𝑀 1
1
=
.
+
𝑣 𝑉𝑚𝑎𝑥 𝑆
𝑉𝑚𝑎𝑥
Graph of the equation is a linear dependence of reciprocal value of measured absorbance 1/A to
reciprocal value of concentration of lactate 1/[S]. Intersection with axis y is 1/Vmax and intersection with
axis x is -1/KM.
1/A
1/Vmax
1/[S]
-1/KM
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APPENDIX 2: MATERIAL SAFETY DATA SHEET
1. Sodium hydroxide
Risk phrases:
H314 Causes burns.
Safety phrases:
P280 Wear suitable protective clothing, gloves and eye/face protection.
2. 2,4-dinitrophenylhydrazine
Risk phrases:
H228 Flammable solid.
H302 Harmful if swallowed.
H315 Irritating to skin.
H319 Causes eye irritation.
EUH001 Explosive if dry.
Safety phrases:
Protect from heat/sparks/flames/hot surface. – Smoking is not allowed.
P280 Wear suitable protective clothing, gloves and eye/face protection.
3. TRIS - Tris(hydroxymethyl)aminomethan
Risk phrases:
H319 Causes eye irritation.
H335 Irritating to respiratory system.
H315 Irritating to skin.
Safety phrases:
P261 Prevent inhalation of dust.
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